Lancaster, Johnathan M.

Abstract Mi(cro)RNAs are short non-coding RNA molecules that play a key role in carcinogenesis by regulating tumor suppressors and oncogenes. Published data has implicated miRNAs in ovarian cancer (OC) development and progression, and we hypothesize that single nucleotide polymorphisms (SNPs) in sites of miRNA: messenger RNA (mRNA) binding may influence OC risk by altering expression levels of targeted mRNAs. To evaluate associations between SNPs in miRNA binding sites and OC risk, we used bioinformatics tools and public databases to identify miRNA binding SNPs relevant to ovarian cancer. We then evaluated the frequency of approximately 1,100 SNPs in ∼20,000 epithelial OC cases and 20,000 controls represented in the international Collaborative Oncological Gene-environment Study. Unconditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) between genotypes and case status, with adjustment for the first five principal components representing European ancestry. Log-additive genetic models were applied to each SNP, modeling the number of copies of the minor allele. Subgroup analysis was conducted for serous adenocarcinomas, the most predominant histologic subtype of epithelial OC. Preliminary analysis of 11,215 invasive epithelial OCs and 15,910 controls included four SNPs with P<10−6 and 1 SNP with P<10−5. Analysis of 6,215 invasive serous adenocarcinomas revealed two SNPs with P<10−7 and three SNPs with P<10−6. Noteworthy is rs6104808, a C>T SNP predicted to reside in a highly conserved miRNA binding site of BTBD3 (BTB POZ domain containing 3), a transcription factor suggested to be involved in cell proliferation, cancer progression, and response to platinum-based chemotherapy. rs6104808 was associated with a decreased risk of OC overall (OR (95% CI): 0.42 (0.28-0.62), P=1.26 x 10−5)), and the association was stronger among cases with serous adenocarcinoma (OR (95% CI): 0.30 (0.17-0.50), P=9.89 x 10−6)). This represents the largest, most comprehensive epidemiologic study to date to evaluate associations between miRNA binding site SNPs and OC susceptibility. Although based on a preliminary analysis, these data suggest a significant role and putative biological mechanism for common germline variants in miRNA binding sites leading to cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2927. doi:1538-7445.AM2012-2927

Abstract Background: Single nucleotide polymorphisms (SNP) in microRNA-related genes have been associated with epithelial ovarian cancer (EOC) risk in two reports, yet associated alleles may be inconsistent across studies. Methods: We conducted a pooled analysis of previously identified SNPs by combining genotype data from 3,973 invasive EOC cases and 3,276 controls from the Ovarian Cancer Association Consortium. We also conducted imputation to obtain dense coverage of genes and comparable genotype data for all studies. In total, 226 SNPs within 15 kb of 4 miRNA biogenesis genes (DDX20, DROSHA, GEMIN4, and XPO5) and 23 SNPs located within putative miRNA binding sites of 6 genes (CAV1, COL18A1, E2F2, IL1R1, KRAS, and UGT2A3) were genotyped or imputed and analyzed in the entire dataset. Results: After adjustment for European ancestry, no overall association was observed between any of the analyzed SNPs and EOC risk. Conclusions: Common variants in these evaluated genes do not seem to be strongly associated with EOC risk. Impact: This analysis suggests earlier associations between EOC risk and SNPs in these genes may have been chance findings, possibly confounded by population admixture. To more adequately evaluate the relationship between genetic variants and cancer risk, large sample sizes are needed, adjustment for population stratification should be carried out, and use of imputed SNP data should be considered. Cancer Epidemiol Biomarkers Prev; 20(8); 1793–7. ©2011 AACR.

Abstract Background: Mitochondria contribute to oxidative stress, a phenomenon implicated in ovarian carcinogenesis. We hypothesized that inherited variants in mitochondrial-related genes influence epithelial ovarian cancer (EOC) susceptibility. Methods: Through a multicenter study of 1,815 Caucasian EOC cases and 1,900 controls, we investigated associations between EOC risk and 128 single nucleotide polymorphisms (SNPs) from 22 genes/regions within the mitochondrial genome (mtDNA) and 2,839 nuclear-encoded SNPs localized to 138 genes involved in mitochondrial biogenesis (BIO, n = 35), steroid hormone metabolism (HOR, n = 13), and oxidative phosphorylation (OXP, n = 90) pathways. Unconditional logistic regression was used to estimate OR and 95% CI between genotype and case status. Overall significance of each gene and pathway was evaluated by using Fisher's method to combine SNP-level evidence. At the SNP level, we investigated whether lifetime ovulation, hormone replacement therapy (HRT), and cigarette smoking were confounders or modifiers of associations. Results: Interindividual variation involving BIO was most strongly associated with EOC risk (empirical P = 0.050), especially for NRF1, MTERF, PPARGC1A, ESRRA, and CAMK2D. Several SNP-level associations strengthened after adjustment for nongenetic factors, particularly for MTERF. Statistical interactions with cigarette smoking and HRT use were observed with MTERF and CAMK2D SNPs, respectively. Overall variation within mtDNA, HOR, and OXP was not statistically significant (empirical P > 0.10). Conclusion: We provide novel evidence to suggest that variants in mitochondrial biogenesis genes may influence EOC susceptibility. Impact: A deeper understanding of the complex mechanisms implicated in mitochondrial biogenesis and oxidative stress may aid in developing strategies to reduce morbidity and mortality from EOC. Cancer Epidemiol Biomarkers Prev; 20(6); 1131–45. ©2011 AACR.

Abstract Defective microRNA (miRNA) biogenesis contributes to the development and progression of epithelial ovarian cancer (EOC). In this study, we examined the hypothesis that single nucleotide polymorphisms (SNP) in miRNA biogenesis genes may influence EOC risk. In an initial investigation, 318 SNPs in 18 genes were evaluated among 1,815 EOC cases and 1,900 controls, followed up by a replicative joint meta-analysis of data from an additional 2,172 cases and 3,052 controls. Of 23 SNPs from 9 genes associated with risk (empirical P < 0.05) in the initial investigation, the meta-analysis replicated 6 SNPs from the DROSHA, FMR1, LIN28, and LIN28B genes, including rs12194974 (G>A), an SNP in a putative transcription factor binding site in the LIN28B promoter region (summary OR = 0.90, 95% CI: 0.82–0.98; P = 0.015) which has been recently implicated in age of menarche and other phenotypes. Consistent with reports that LIN28B overexpression in EOC contributes to tumorigenesis by repressing tumor suppressor let-7 expression, we provide data from luciferase reporter assays and quantitative RT-PCR to suggest that the inverse association among rs12194974 A allele carriers may be because of reduced LIN28B expression. Our findings suggest that variants in LIN28B and possibly other miRNA biogenesis genes may influence EOC susceptibility. Cancer Res; 71(11); 3896–903. ©2011 AACR.

Abstract Mitochondria have been implicated in carcinogenesis because of their central role in apoptosis, free radical production, and cellular energy metabolism. Although studies have identified somatic mitochondrial DNA mutations in ovarian cancers, little attention has been directed to the possible role of germline mitochondrial variants in mitochondrial dysfunction and subsequent ovarian cancer development. Through an ongoing epithelial ovarian cancer genome-wide association study of 1,856 invasive cases (59% with serous carcinomas) and 1,912 controls (frequency-matched on age, race, and study site), we genotyped 138 SNPs tagging variation in the maternally-inherited mitochondrial genome (mtDNA) to evaluate the hypothesis that single nucleotide polymorphisms (SNPs) in mtDNA are associated with ovarian cancer risk. All subjects were non-Hispanic, non-Jewish Caucasians, and were genotyped with the Illumina 610-quad Beadchip. Samples and SNPs with call rates less than 95% were excluded. Subjects with ambiguous gender, unresolved identical genotypes, and less than 80% European ancestry were excluded. For each SNP, logistic regression was used to estimate odds ratios (OR) and corresponding 95% confidence intervals (CI) between carriers of the minor versus major maternally-inherited allele and case status. Subgroup analysis was conducted to estimate allele-specific risks between serous-only cases and controls. Haplotype analysis was performed for regions containing statistically significant SNPs (p<0.05). Two rare SNPs, C16329T in the displacement loop gene (MT-D-loop) (OR: 6.72, 95%CI: 1.5-29.8, p=0.004; minor allele frequency (MAF)=0.001) and C15905T in the tRNA threonine gene (MT-TT) (OR: 1.60, 95%CI: 1.1-2.3, p=0.009; MAF=0.027), were associated with increased ovarian cancer risk; whereas T6777C in the cytochrome c oxidase subunit 1 gene (MT-CO1) (OR: 0.68, 95%CI: 0.51-0.91, p=0.009; MAF=0.063) was associated with decreased risk. All three SNPs remained statistically significant after adjustment for multiple comparisons within the mitochondrial SNPs using permutation testing. C16329T and T6777C were also significantly associated among cases with serous histology, with ORs (95% CIs) of 6.13 (1.2-29.6) and 0.61 (0.43-0.87), respectively. Haplotype analysis for each of the three regions investigated revealed significant haplotype-disease associations; however, findings suggest that the respective associations were driven by the most significant SNP(s) listed above. This is the first large-scale epidemiologic study to provide evidence that SNPs in mitochondrial genes may be novel risk factors for ovarian cancer. Future investigation of additional mitochondrial variants (i.e. nuclear-encoded mitochondrial proteins) is necessary to more comprehensively investigate this association. Furthermore, replication of the most promising SNPs in a larger population is warranted to validate these findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2835.